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Effect of the Freezing Step in the Stability and Bioactivity of Protein-Loaded PLGA Nanoparticles Upon Lyophilization

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Abstract

Purpose

The freezing step in lyophilization is the most determinant for the quality of biopharmaceutics. Using insulin as model of therapeutic protein, our aim was to evaluate the freezing effect in the stability and bioactivity of insulin-loaded PLGA nanoparticles. The performance of trehalose, sucrose and sorbitol as cryoprotectants was evaluated.

Methods

Cryoprotectants were co-encapsulated with insulin into PLGA nanoparticles and lyophilized using an optimized cycle with freezing at −80°C, in liquid nitrogen, or ramped cooling at −40°C. Upon lyophilization, the stability of protein structure and in vivo bioactivity were assessed.

Results

Insulin was co-encapsulated with cryoprotectants resulting in particles of 243–394 nm, zeta potential of −32 to −35 mV, and an association efficiency above 90%. The cryoprotectants were crucial to mitigate the freezing stresses and better stabilize the protein. The insulin structure maintenance was evident and close to 90%. Trehalose co-encapsulated insulin-loaded PLGA nanoparticles demonstrated enhanced hypoglycemic effect, comparatively to nanoparticles without cryoprotectant and added with trehalose, due to a superior insulin stabilization and bioactivity.

Conclusions

The freezing process may be detrimental to the structure of protein loaded into nanoparticles, with negative consequences to bioactivity. The co-encapsulation of cryoprotectants mitigated the freezing stresses with benefits to protein bioactivity.

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Abbreviations

AAC:

Area above the curve

AE:

Association efficiency

ATR-FTIR:

Attenuated Total Reflectance-Fourier transform infrared spectroscopy

CD:

Circular dichroism

DSC:

Differential scanning calorimetry

HPLC:

High performance liquid chromatography

LC:

Loading capacity

PA:

Pharmacological availability

PLGA:

Poly(lactic-co-glycolic acid)

PVA:

Polyvinyl alcohol

RE:

Retention efficiency

SEM:

Scanning electron microscopy

Tc:

Collapse temperature

Tg:

Glass transition temperature

Tg’:

Glass transition temperature of the frozen sample

XRPD:

X-Ray powder diffraction

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ACKNOWLEDGMENTS AND DISCLOSURES

Pedro Fonte and Fernanda Andrade would like to thank Fundação para a Ciência e a Tecnologia (FCT), Portugal (PTDC/SAL-FCT/104492/2008; SFRH/BD/78127/2011) and (SFRH/BD/73062/2010) for financial support. This work was also financed by European Regional Development Fund (ERDF) through the Programa Operacional Factores de Competitividade − COMPETE, by Portuguese funds through FCT in the framework of the project PEst-C/SAU/LA0002/2013, and cofinanced by North Portugal Regional Operational Programme (ON.2 − O Novo Norte) in the framework of Project SAESCTN-PIIC&DT/2011 under the National Strategic Reference Framework (NSRF). It is also acknowledged the financial support from FCT/MEC through national funds and co-financed by FEDER, under the Partnership Agreement PT2020 (UID/MULTI/04378/2013 - POCI/01/0145/FERDER/007728). Abbot Laboratories, Portugal is acknowledged for kindly provide the Precision Xtra® blood glucose meter and test strips. The authors also thank to Dorthe Ørbæk from the Faculty of Health and Medical Sciences, University of Copenhagen, for the XRPD experiments.

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Authors and Affiliations

  1. UCIBIO, REQUIMTE, Department of Chemical Sciences - Applied Chemistry Lab, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050-113, Porto, Portugal

    Pedro Fonte & Salette Reis

  2. CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde, Rua Central de Gandra 1317, 4585-116, Gandra PRD, Portugal

    Pedro Fonte, Vítor Seabra & Bruno Sarmento

  3. Laboratory of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050-113, Porto, Portugal

    Fernanda Andrade

  4. IBEC, Institute for Bioengineering of Catalonia, 08028, Barcelona, Spain

    Fernanda Andrade

  5. School of Pharmacy, University of Barcelona, 08028, Barcelona, Spain

    Fernanda Andrade

  6. i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal

    Cláudia Azevedo & Bruno Sarmento

  7. INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Rua Alfredo Allen 208, 4200-135, Porto, Portugal

    Cláudia Azevedo & Bruno Sarmento

  8. iMed. ULisboa, Department of Galenic Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Lisbon, Avenida Professor Gama Pinto, 1649-003, Lisbon, Portugal

    João Pinto

  9. Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, 2100, Copenhagen, Denmark

    Marco van de Weert

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  1. Pedro Fonte
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  2. Fernanda Andrade
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  3. Cláudia Azevedo
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Correspondence to Pedro Fonte or Bruno Sarmento.

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Fonte, P., Andrade, F., Azevedo, C. et al. Effect of the Freezing Step in the Stability and Bioactivity of Protein-Loaded PLGA Nanoparticles Upon Lyophilization. Pharm Res 33, 2777–2793 (2016). https://doi.org/10.1007/s11095-016-2004-3

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